Platelet Drp1 phosphorylation provides a platform for immune-based platelet function testing

Abstract

Abstract Dynamin-related protein 1 (Drp1) is an abundant platelet protein best known for its function in mitochondrial fission. However, little is known about how Drp1 is controlled during platelet activation. While evaluating signaling pathways leading to phosphorylation of Drp1 in platelets, we identified a phosphorylation network wherein activation of G protein–coupled receptors or immunoreceptor tyrosine-based activation motif (ITAM)/hemITAM receptors resulted in phosphorylation of Ser616-Drp1 via p38. These signaling mechanisms were reinforced by ADP- and thromboxane A2 (TxA2)-mediated amplification pathways. In contrast, exposure of platelets to pacifying agents such as prostaglandin E1 or nitric oxide resulted in Ser637-Drp1 phosphorylation by cyclic nucleotide-dependent protein kinases. This unique circuitry was leveraged to develop immune-based platelet function assays, enabling enzyme-linked immunosorbent assay and lateral flow assay formats. Compared with standard platelet function assays, Drp1 phosphorylation remained robust in whole blood samples following agitation or extended incubation, and samples could be frozen for batching. As proof of principle for antiplatelet testing, phospho-Drp1 measurements were obtained at baseline, during a week of aspirin or clopidogrel exposure, and during a week of washout. Arachidonic acid–induced Ser616-Drp1 phosphorylation following aspirin ingestion demonstrated an enhanced dynamic range with improved linearity relative to light transmission aggregometry and an improved signal-to-noise ratio relative to the VerifyNow aspirin test. Ser637-Drp1 phosphorylation enabled sensitive detection of clopidogrel ingestion. These studies elucidate the unique signaling circuit controlling Drp1 phosphorylation in platelets and validate the approach of using detailed knowledge of platelet signaling pathways to develop high-fidelity immune-based assays to monitor platelet function.